This project centers on our recent discovery of separate sources of intracellular calcium for synchronous and asynchronous modes of synaptic transmission at the zebrafish neuromuscular junction. Asynchronous release, in particular, has received much attention, as a result of its newly appreciated role in synaptic plasticity. However, the mechanisms causal to preferential release through the asynchronous mode is presently a hotly contested subject. Our lab was the first to identify a calcium sensor specific to asynchronous release, and we now find that, additionally, synchronous versus asynchronous release is mediated by two distinct voltage- activated calcium channel isoforms. The synchronous release utilizes a P/Q type calcium channel and the asynchronous release utilizes a voltage dependent calcium channel isoform that awaits molecular identification through Aim 1 experiments. In this proposal, we present much unpublished data in support of differential locations of these two channel isoforms, with the synchronous channel in the synaptic bouton and the asynchronous isoform located extrasynaptically at axonal branch points. Establishing its location in the cell forms the basis of aim 2 experiments. Additionally, the combined technologies of in vivo calcium imaging, exocytosis indicator lines, and paired recordings have pointed to activation of a calcium wave that is activated by the asynchronous calcium channel isoform and propagates through active release of internal calcium to reach the synapse. This is causal to the signature delayed onset and persistence of asynchronous release at nearly all studied synapses. Numerous reports of calcium waves exist for both the neuromuscular junction and central neurons but, until now, the physiological significance vis-a-vis synaptic transmission has remained obscure. In aim 2 we will determine the molecular basis of calcium release through activation of the extrasynaptic calcium current establish the links to the asynchronous release process. Finally, in Aim 3 we will use the collective advantages of zebrafish to test the requirement for each proposed signaling molecule in the physiology of spontaneous, synchronous and asynchronous release modes.